PROJECT SUMMARY The goal this Phase 1 SBIR project is to develop an agonist bias assay for screening drugs that target seven transmembrane G-protein coupled receptors (GPCRs). Phase 1 will establish the feasibility of a robust assay platform to detect when particular GPCR agonists activate both ?-arrestin and G-protein pathways. This assay will detect responses in living cells on automated fluorescence plate readers, and will be compatible with screening in any cell type including neurons, pancreatic islets, cardiomyocytes, transformed cell lines and iPSC-derived cells. Depending upon the GPCR and cell type, biased agonism can be therapeutically beneficial or produce unwanted or harmful side effects (Violin and Lefkowitz, 2007). Quantifying bias in GPCR signaling could help to identify treatments that avoid adverse effects. Detecting agonist bias early in the screening process, in cell types that are relevant to disease, has the potential to reduce the risk and cost of drug discovery. The following specific aims will establish the feasibility of this assay: Aim 1: Develop dimerization-dependent fluorescent proteins to report ?-arrestin protein interactions involved in signaling. The dimerization-dependent fluorescent protein (ddFP) sensors will serve as a rapid prototype to be used as a standard for verifying sensors described in Aim 2. The ddFP sensors will be a functional readout for the protein interactions characteristic of ?-arrestin signaling. The ddFP sensors will be built using standard molecular cloning in the matter of a few weeks and will be detectable using epi- fluorescence microscopy. These prototype sensors are not amenable for detection on automated plate readers but will be useful for lower throughput, nonquantitative applications. Aim 2: Develop ?-arrestin signaling sensors that are based on one, bright fluorescent protein that have such large signals that they are detectable on an automated fluorescence plate reader. Building on the ?-arrestin protein interactions in aim one, we will harness the large scale movements of ?-arrestin associated proteins to create a bright green sensor with circularly permuted mNeonGreen. Libraries of candidate sensors will be built and screened using an iterative, directed evolution approach. Aim 3: Test a fluorescence plate reader assay that simultaneously detects G-protein and ?-arrestin signaling typical of biased agonism. ?2-adrenergic receptor activation by agonists that signal through either or both the Gs pathway and the ?-arrestin pathway (Drake et al., 2008) will be used to test the assay on a standard fluorescence plate reader. Responses from prototype sensors for ?-arrestin signaling, developed in aims one and two, and a previously developed red cAMP sensor (Tewson et al., 2016) will be compared to determine levels of agonist bias from known ?2-adrenergic agonists. www.montanamolecular.com